Project D: Hypocretin and Histamine studies in the Zebrafish Hypocretin and histamine are now known to be important arousal signals. Anatomical correlates c hypocretin, histamine, and other cell groups relevant to sleep regulation, as well as the cognate homologou genes, have been identified in zebrafish and appear to anatomically interconnect similarly as in mammals We have identified a sleep-like state in zebrafish and can objectively characterize alterations in this behavio with an automated digital video system. The goal of this revised proposal is to take advantage of th attributes of the zebrafish as a genetic model organism, to develop a better understanding of factors relevan to sleep regulation in humans. Specifically, we will validate the zebrafish as a model for the study of sleep disturbances in relationship to hypocretin and histamine through the use of transgenic and gene-targeted zebrafish by characterization of the behavioral effects of loss of, or overexpression of hypocretin (HCRT) o histidine decarboxylase (HOC) and loss of expression of their relevant receptors (hypocretin recepto (HCRTR), histamine H1 receptor (H1). We hypothesize that animals deficient in hypocretinergic o listaminergic transmission will have altered rest/activity patterns. These hypothesized altered rest/activity patterns will demonstrate functional conservation of these systems in the zebrafish, and validate the use o zebrafish as a model in the study of study of sleep disturbances in relationship to hypocretin and histamine, We will also exploit the forward genetics advantages of the zebrafish to identify novel developmental genes affecting the hypocretin and histamine systems by screening for ENU-induced mutations resulting in loss of expression of HCRT, HOC and their receptors, or abnormal placement of these cells. Such novel genes may be relevant to the development of narcolepsy and hypersomnia in humans. We will also characterize these dentified mutants in terms of cellular specificity, and gene expression profiles, and behavior as a preliminary step toward positional cloning. Finally, we will genetically map the most interesting mutations (those with cell specificity and/or robust behavioral phenotype). These studies will be valuable in our understanding of the etiology of narcolepsy and hypersomnia.